Validation of internal control genes for expression studies: Effects of the neurotrophin BDNF on hippocampal neurons

Santos, Ana Rita

doi:10.1002/jnr.21796

Cited by 55 publications

(62 citation statements)

References 40 publications

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“…Analysis was performed using the ∆∆ C t method, where relative quantity (RQ) = 2 -∆∆Ct and ∆∆Ct = ∆C t , sample -∆C t , reference. ∆C t , sample is the difference in threshold cycle between any developmental stage or brain region and the endogenous reference gene hypoxanthine phosphoribosyl transferase (HPRT) [21] and ∆C t , reference is the difference in threshold cycle between the reference sample/calibrator (adult brain or cerebral cortex for the developmental expression or adult brain expression profiles, respectively) and the endogenous control (HPRT). Santos and Duarte found HPRT to be the most reliable reference gene for real-time PCR [21].…”

Section: Quantitative Real Time Pcrmentioning

confidence: 99%

“…∆C t , sample is the difference in threshold cycle between any developmental stage or brain region and the endogenous reference gene hypoxanthine phosphoribosyl transferase (HPRT) [21] and ∆C t , reference is the difference in threshold cycle between the reference sample/calibrator (adult brain or cerebral cortex for the developmental expression or adult brain expression profiles, respectively) and the endogenous control (HPRT). Santos and Duarte found HPRT to be the most reliable reference gene for real-time PCR [21]. HPRT expression reflects the average expression of multiple common reference genes, making it the best choice when using only a single normalization gene [22].…”

Section: Quantitative Real Time Pcrmentioning

confidence: 99%

“…HPRT expression reflects the average expression of multiple common reference genes, making it the best choice when using only a single normalization gene [22]. HPRT has also been shown to be expressed at constant levels compared to other reference genes [21,[23][24][25]. To determine the stability of HPRT in all developmental stages used, its expression was validated against additional commonly used reference genes, including B2m (beta-2-microglobulin), Gapdh (glyceraldehyde 3-phosphate dehydrogenase), Gusb (beta-glucuronidase), Tfrc (transferrin receptor), and Pgk1 (phosphoglycerate kinase 1) [21,26].…”

Section: Quantitative Real Time Pcrmentioning

confidence: 99%

“…HPRT has also been shown to be expressed at constant levels compared to other reference genes [21,[23][24][25]. To determine the stability of HPRT in all developmental stages used, its expression was validated against additional commonly used reference genes, including B2m (beta-2-microglobulin), Gapdh (glyceraldehyde 3-phosphate dehydrogenase), Gusb (beta-glucuronidase), Tfrc (transferrin receptor), and Pgk1 (phosphoglycerate kinase 1) [21,26]. The Ct value of the six genes was analyzed for validation of expression value (M) across development using the geNorm program [27].…”

Section: Quantitative Real Time Pcrmentioning

confidence: 99%

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Developmental expression of P5 ATPase mRNA in the mouse

Weingarten¹,

Dave²,

Li³

et al. 2012

Cellular and Molecular Biology Letters

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P5 ATPases (ATP13A1 through ATP13A5) are found in all eukaryotes. They are currently poorly characterized and have unknown substrate specificity. Recent evidence has linked two P5 ATPases to diseases of the nervous system, suggesting possible importance of these proteins within the nervous system. In this study we determined the relative expression of mouse P5 ATPases in development using quantitative real time PCR. We have shown that ATP13A1 and ATP13A2 were both expressed similarly during development, with the highest expression levels at the peak of neurogenesis. ATP13A3 was expressed highly during organogenesis with one of its isoforms playing a more predominant role during the period of neuronal development. ATP13A5 was expressed most highly in the adult mouse brain. We also assessed the expression of these genes in various regions of the adult mouse brain. ATP13A1 to ATP13A4 were expressed differentially in the cerebral cortex, hippocampus, brainstem and cerebellum while levels of ATP13A5 were fairly constant between these brain regions. Moreover, we demonstrated expression of the ATP13A4 protein in the corresponding brain regions using immunohistochemistry. In summary, this study furthers our knowledge of P5-type ATPases and their potentially important role in the nervous system.

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Section: Quantitative Real Time Pcrmentioning

confidence: 99%

Section: Quantitative Real Time Pcrmentioning

confidence: 99%

Section: Quantitative Real Time Pcrmentioning

confidence: 99%

Section: Quantitative Real Time Pcrmentioning

confidence: 99%

See 2 more Smart Citations

Developmental expression of P5 ATPase mRNA in the mouse

Weingarten¹,

Dave²,

Li³

et al. 2012

Cellular and Molecular Biology Letters

View full text Add to dashboard Cite

show abstract

“…GAPDH, 18S or CycA (Abildayeva et al, 2006;Karten et al, 2006;Liang et al, 2004). Recently, suitable reference genes were described for rat brain tissue and hippocampal neurons under different experimental conditions (Bonefeld et al, 2008;Langnaese et al, 2008;Santos and Duarte, 2008). However, to our knowledge no study has yet been performed on glial cells.…”

Section: Introductionmentioning

confidence: 99%

Selection of reference genes for gene expression studies in rat oligodendrocytes using quantitative real time PCR

Nelissen

Smeets

Mulder

et al. 2010

Journal of Neuroscience Methods

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Quantitative real time polymerase chain reaction (qPCR) has become a widely used tool to examine gene expression levels. Reliable quantification, however, depends on a proper normalization strategy. Normalization with multiple reference genes is becoming the standard, although the most suitable reference genes depend on the applied treatment as well as the tissue or cell type studied. In this study the stability of various reference genes was investigated in cultures of oligodendrocytes derived from either mature or neonatal rats, the latter also in the presence of the liver X receptor (LXR) agonist.The expression stability of ten commonly used reference genes (HPRT, GAPDH, 18S, ActB, CycA, Tbp, Rpl13A, YWHAZ, HMBS, Pgk1) was analyzed using geNorm and NormFinder.When comparing the different types of cell cultures, Rpl13A, CycA, Pgk1 and YWHAZ were identified as most stable genes. After LXR agonist treatment, CycA, Pgk1 and Rpl13A were found to be the most stable by both geNorm and NormFinder. HMBS and the commonly used housekeeping genes GAPDH and 18S turned out to be the most variable according to geNorm and NormFinder. In conclusion, the use of multiple reference genes, instead of only one, in qPCR experiments with rat oligodendrocytes is strongly advised and standard housekeeping genes such as GAPDH and 18S are not recommended as they appear to be relatively unstable under the experimental conditions used. Reference gene selection should always be performed for each individual experiment, since useful reference genes are very specific for every situation.

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Selection of reference genes for real‐time quantitative reverse transcription‐polymerase chain reaction in hippocampal structure in a murine model of temporal lobe epilepsy with focal seizures

Pernot¹,

Dorandeu²,

Beaup³

et al. 2009

J of Neuroscience Research

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Reference genes are often used to normalize expression of data from real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and only a validation of their stability during a given experimental paradigm leads to reliable interpretations. The present study was thus designed to validate potential reference genes in a mouse model of mesiotemporal lobe epilepsy (MTLE) with focal seizures after unilateral intrahippocampal injection of kainate (KA). Ipsilateral and contralateral hippocampi were removed during nonconvulsive status epilepticus (5 hr), epileptogenesis (7 days), and the chronic period of recurrent focal seizures (21 days). Naive animals were equally studied. The stability of eight potential reference genes (hypoxanthine phosphoribosyltransferase, Hprt1; peptidylprolyl isomerase A, Ppia; TATA box binding protein, Tbp; beta-actin, Actb; acidic ribosomal phosphoprotein P0, Arbp; glyceraldehyde-3-phosphate dehydrogenase, Gapdh; ribosomal RNA 18S, 18S rRNA; and glucuronidase beta, Gusb) were determined using geNorm and NormFinder software. The first five (Hprt1, Ppia, Tbp, Actb, and Arbp) were found to be stable across the different phases of the disease and appeared adequate for normalizing RT-qPCR data in this model. This was in contrast to the other three (18S rRNA, Gapdh, and Gusb), which showed unstable expressions and should be avoided. The analysis of KA-induced changes in the expression of glial fibrillary acidic protein (Gfap) gene resulted in various relative expressions or even a completely different pattern when unstable reference genes were used. These results highlight the absolute need to validate the reference genes for a correct interpretation of mRNA quantification.

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Validation of internal control genes for expression studies: Effects of the neurotrophin BDNF on hippocampal neurons

Cited by 55 publications

References 40 publications

Developmental expression of P5 ATPase mRNA in the mouse

Developmental expression of P5 ATPase mRNA in the mouse

Selection of reference genes for gene expression studies in rat oligodendrocytes using quantitative real time PCR

Selection of reference genes for real‐time quantitative reverse transcription‐polymerase chain reaction in hippocampal structure in a murine model of temporal lobe epilepsy with focal seizures

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